(Acta Paediatr Jon 1992; 34: 636
-
641)
Pulmonary Surfactant Apoprotein-A in Neonates with Different Respiratory Disorders Hiroshi Nagai", M.D., Yunosuke Ogawa, M.D., Hideshi Eguchi, M.D., Kenji Kamiya, M.D., Norihisa Koyama, M.D., Taihei Tanaka, M.D., Jiro Takasaki, M.D., Eiko Takada, M.D., Yoichi Ohama, M.D., Koji Kaneko, M.D. and Naoko Yoshida, M.D. Department of Pediatrics, Saitama Medical Center, Saitama Medical School, Kawagoe, Saitama, Japan A serial determination of pulmonary surfactant apoprotein-A (SP-A) was made on tracheal aspirates from seven intubated infants with different types of respiratory failure in the first week of life. A two-site immunoassay with monoclonal antibodies was adopted to determine the SP-A concentration. The concentrations of albumin in the same samples were also assayed, and these data were expressed as the ratio of SP-A to albumin (SP-A/albumin ratio), and evaluated against clinical data such as the arterial-alveolar oxygen tension ratio (a/APo2) or ventilatory index. In infants with respiratory distress syndrome, the SP-A/ albumin ratio was initially low, and increased gradually in the first few days of life with the improvement of a/APo2 and ventilatory index. The complication of pulmonary hemorrhage due to patent ductus arteriosus (PDA) resulted in a temporary decrease in the ratio. The infant with transient tachypnea of the newborn showed higher concentration from the first day of life and, in the course of PDA without pulmonary hemorrhage, the ratio did not decrease. The cases of congenital pneumonia showed the SP-A/albumin ratio remaining low while the infection was evident. These data suggest that the SP-Malbumin ratio of the tracheal aspirate can be used for the quantitative and qualitative evaluation of endogenous pulmonary surfactant in newborn infants with different respiratory disorders.
Key Words Surfactantapoprotein-A, Newborn infant, Arterial-alveolar oxygen tension ratio, Ventilatory index, Tracheal aspirate
introduction Pulmonary surfactant contains several specific apoproteins, and four families of these apoproteins SP-A, B, C and D have been identified Received July 9. I992 Correspondence address: Yunosuke Ogawa, M.D., Department of Pediatrics, Saitama Medical Center, Saitama Medical School, 198 1 Kamoda-Tsujidou, Kawagoe, Saitama 350, Japan. *Deceased as at April 12. 1992.
[ 1,2]. SP-A is the major surfactant apoprotein and its concentration in amniotic fluid, tracheal aspirate or gastric aspirate of the newborn infant is useful for the evaluation of fetal lung maturity or the prediction of respiratory distress syndrome (RDS) of the newborn infant [3-5]. The pathogenesis of RDS is recognized as a deficiency of pulmonary surfactant, mainly due to lung immaturity. In addition, interference of surfactant function caused by inhibitors such
637 (59) Nagai et al. as meconium aspirated to the lung or proteins leaked from sera can develop similar pathophysiology caused by this quantitative deficiency. Recently, the effectiveness of exogenous pulmonary surfactant administration has been reported in cases without RDS whose respiratory failure might be due to insufficient endogenous pulmonary surfactant [61. However, little information is available about the dynamic change of endogenous pulmonary surfactant in such disorders. The aim of this study was to investigate the dynamic change of endogenous pulmonary surfactant in respiratory disorders other than classic RDS in the first week of life, and to compare the SP-A concentration of tracheal aspirate with clinical and laboratory findings.
Materials and Methods Patients and specimens A total of 30 samples were obtained from one full-term and six preterm infants who were admitted to the neonatal intensive care unit at Saitama Medical Center, Saitama Medical School, from February to December 1990. The diagnoses of these infants were as follows: pure RDS (one) and complicated RDS (three, all of whom received exogenous pulmonary surfactant therapy with surfactant-TA), transient tachypnea of the newborn (TTN; one) and congenital pneumonia (two; Table 1). All patients received mechanical ventilation soon after birth. Three of these infants were treated with conventional mechanical ventilation (CMV) and the other four infants were initially on high frequency oscillatory ventilation (HFOV), accord-
ing to the judgment of the attending doctor. The initial samples were obtained within the first 24 hr of life and subsequently every 24 hr after the initial sample was obtained. All samples were frozen at - 80°C until assayed. Parental consent was obtained for collecting all samples.
SP-A assay The two-site immunoassay with two monoclonal antibodies described by Kuroki et a1 was adopted to determine the SP-A concentration [3, 71. The tracheal aspirate sample or SP-A standard was mixed with peroxidase-labeled monoclonal antibody (PE-I0) in the test tube, and then the second antibody (PC-6) immobilized on polystyrene beads was placed in each test tube. After incubation at 37°C for 90 min, the test tube was washed, and the substrate (0.005 mol/l H,O, in 0.1 mol/l phosphate/ citrate buffer, pH 4.0) and developer solution (0.06% tetramethyl benzidine-HCl, pH 2.0) were added to each tube. After the second incubation at 37°C for 30 min, the peroxidase reaction was stopped by the addition of sulfuric acid. The absorbance was measured at 450 nm. All samples were run in duplicate. The concentration of albumin for each sample was also measured by laser nephrometry. The concentration of SP-A was standardized by dividing the SP-A content by the albumin present in the aspirate. The results were evaluated against clinical condition and laboratory data, such as arterial-alveolar oxygen tension ratio (a/APo2) and ventilatory index (Fio2 MAP/Pao,). +
Table 1. Clinical characteristics of the study group Case
Gestational age
Birthweight (g)
Diagnosis
Mode of ventilation
A B C D
26 weeks, 6 days 26 weeks, 5 days 26 weeks, 3 days 33 weeks, 5 days
1020 750 836 1278
CMV HFOV + CMV CMV + HFOV HFOV + CMV
E F G
33 weeks, 3 days 24 weeks, 1 days 38 weeks, 5 days
1228 618 2758
RDS (IV) RDS (IV) t PDA RDS (IV) t PDA RDS (IV) Pena-Shokeir I syndrome TTN + PDA Pneumonia Pneumonia PPHN
CMV CMV HFOV + CMV
Acta Paediatr Jpn
Surfactant apoprotein in tracheal aspirate (60)638
Results The SP-Nalbumin ratios in two preterm infants diagnosed with RDS by clinical features and chest X-ray findings are presented in Fig. 1. The microbubble stability test [8j performed with gastric aspirate obtained at birth was 'weak' in both cases, and the concentrations of SP-A in the same specimens were 0.1 &ml in case A and 0.3 &mi in case B, respectively. The SP-A/ albumin ratios of initial tracheal aspirates were low in both cases (0.1 @mg in case A and 2.7 pglmg in case B, respectively). In case A, the SP-Alalbumin ratio rose to 12.5pgImg on the second day of life along with improvements in the a/AP02 and ventilatory index. In case B, HFOV improved his oxygenation without surfactant replacement therapy and the SP-A/ albumin ratio increased to 21.0pg/mg on the second day of life. However, his condition again deteriorated with the white-out appearance of
chest X-ray. Surfactant-TA was given immediately with the change in the mode of mechanical ventilation from HFOV to CMV. Subsequently, pulmonary hemorrhage due to patent ductus arteriosus (PDA) was confirmed by echocardiography and a temporary decrease in aIAPo2 and the SP-Nalbumin ratio was observed. Single use of mefenamic acid was effective for the closure of the ductus, and the SP-Alalbumin ratio increased again along with the improvement in a/APOZon the fifth day of life. In contrast, in two cases with RDS the elevation of the SP-Nalbumin ratio was not observed (Fig. 2). In case C , the complication of severe pulmonary hemorrhage due to PDA was evident on the second day of life. Switching the ventilation mode from CMV to HFOV improved a/AP02 and the ventilatory index, but the SP-A/ albumin ratio remained less than 5 .Opglmg. Case D had RDS accompanied by Pena-Shokeir I syndrome. She commenced HFOV followed by
-5
A . G A 2 6 w 6 d . B W 1020g,RDS(IV)
5
.D
C GA 25 w
3 d. BW 836 g. RDS ( I V ) + PDA HFOV
CMV
'
S-TA
-t
P0A.rPdmon.r~
CMV
Wmorrhq.
Age (days) Age (days)
D GA 33 w 5 d. BW 1278 g, RDS (IV)+ Pena-Shokeir I synd
-
B GA 26 w 5 d . BW 750 g , ROS ( I V ) + POA
-c
wov
c
CUV
s-ll
c
.
wov
-.
CMV
//
5
0 12
OM
03
2
02
0
1
2
3
4
5
6
Age (days)
Age (days)
Fig. I :
Sequential changes in SP-Nalbumin ratio (Ua/APo2 ), (0-0) and ventilatory index (A-Q) of the infants with RDS. Case B developed pulmonary hemorrhage due to PDA, and a transient decrease of SP-Nalbumin ratio was observed in the course of PDA. S-TA, surfactant-TA.
Vol. 34 No. 6 December 1992
Fig. 2: Sequential changes in SP-A/albumin ratio, a/APo, and ventilatory index (VI) of the infants with complicated respiratory distress syndrome. Case C was complicated with severe pulmonary hemorrhage on the second day of life and case D had Pena-Shokeir I syndrome. Neither case showed an elevation of SP-Nalbumin ratio in the first 4 days of life.
639 (61) Nagai et al.
the administration of surfactant-TA, but her clinical condition remained unstable because of respiratory tract infection. During the first 4 days of life the SP-Nalbumin ratio remained less than 10.0 pg/mg. A case of transient tachypnea of the newborn is presented in Fig. 3. A systolic ejection murmur was audible on the second day of life with cardiomegaly and pulmonary congestion on the chest X-ray. Doppler echocardiography indicated the presence of a large left-to-right shunt through the ductus arteriosus. He responded to mefenamic acid and was weaned from the respirator on the seventh day of life. The SP-A/ albumin ratio was 23.4 pglmg on admission and increased to 77.3 pglmg on the third day of life. No significant decrease of SP-Nalbumin ratio was detected thereafter. Two infants with congenital pneumonia are presented in Fig. 4. Both were treated with surfactant-TA in the hope of removing the secretion and debris from the respiratory tract. Case F, an extremely low birthweight infant, showed an elevation of the SP-Nalbumin ratio to 34.9 pg/mg on the third day of life along with an improvement in respiratory failure. On the other hand, the term infant (case G) had marked hypoxemia in 100%O2 with an a/APo, of 0.03 and a ventilatory index of 0.870 on admission. Assisted ventilation with HFOV followed by the E : GA 33 w 3 d, BW 1228 g. TTN.PDA
*
CMV
*
F :GA 24 w 1 d. BW 61 8 g. Pneumonia
--
100
?
-.-F z c
50
1
2
3
Age (days)
G : GA 38 w 5 d. BW 2758 g. Pneumonia
a
C""
+ PPHN
//
9.7.
-100
?
F C
50
0
1
2
3
4
5
5 9
6
Age (days)
Fig. 4: Sequential changes of SP-A (0) and albumin concentrations in tracheal aspirate of the patients with congenital pneumonia. In case F, the SP-A/ albumin ratio ( G O ) elevated gradually along with the improvement of respiratory tract infection. The concentration of albumin in case G remained high in the first 3 days of life while the infection was evident. PPHN, persistent pulmonary hypertension of the newborn.
administration of surfactant-TA produced a significant recovery with the improvement of alAPo2 to 0.38 and ventilatory index to 0.034 on the first day of life. However, the SP-A/ albumin ratio remained less than 5.0pgmg during the first 3 days of life.
Discussion Pulmonary surfactant contains approximately 8% by weight of specific apoproteins. Recently, surfactant apoproteins were designated into four surfactant protein families (SP-A, B, C and D). SP-A and D are hvdroDhilic and SP-B and SP-C are hydrophobic. Among these surfactant apoproteins, SP-A is the most abundant with a molecular weight of26-38 kDa, and its localization in type I1 alveolar cells has been confirmed immunologically. Because surfactant-TA used
-
641)
Pulmonary Surfactant Apoprotein-A in Neonates with Different Respiratory Disorders Hiroshi Nagai", M.D., Yunosuke Ogawa, M.D., Hideshi Eguchi, M.D., Kenji Kamiya, M.D., Norihisa Koyama, M.D., Taihei Tanaka, M.D., Jiro Takasaki, M.D., Eiko Takada, M.D., Yoichi Ohama, M.D., Koji Kaneko, M.D. and Naoko Yoshida, M.D. Department of Pediatrics, Saitama Medical Center, Saitama Medical School, Kawagoe, Saitama, Japan A serial determination of pulmonary surfactant apoprotein-A (SP-A) was made on tracheal aspirates from seven intubated infants with different types of respiratory failure in the first week of life. A two-site immunoassay with monoclonal antibodies was adopted to determine the SP-A concentration. The concentrations of albumin in the same samples were also assayed, and these data were expressed as the ratio of SP-A to albumin (SP-A/albumin ratio), and evaluated against clinical data such as the arterial-alveolar oxygen tension ratio (a/APo2) or ventilatory index. In infants with respiratory distress syndrome, the SP-A/ albumin ratio was initially low, and increased gradually in the first few days of life with the improvement of a/APo2 and ventilatory index. The complication of pulmonary hemorrhage due to patent ductus arteriosus (PDA) resulted in a temporary decrease in the ratio. The infant with transient tachypnea of the newborn showed higher concentration from the first day of life and, in the course of PDA without pulmonary hemorrhage, the ratio did not decrease. The cases of congenital pneumonia showed the SP-A/albumin ratio remaining low while the infection was evident. These data suggest that the SP-Malbumin ratio of the tracheal aspirate can be used for the quantitative and qualitative evaluation of endogenous pulmonary surfactant in newborn infants with different respiratory disorders.
Key Words Surfactantapoprotein-A, Newborn infant, Arterial-alveolar oxygen tension ratio, Ventilatory index, Tracheal aspirate
introduction Pulmonary surfactant contains several specific apoproteins, and four families of these apoproteins SP-A, B, C and D have been identified Received July 9. I992 Correspondence address: Yunosuke Ogawa, M.D., Department of Pediatrics, Saitama Medical Center, Saitama Medical School, 198 1 Kamoda-Tsujidou, Kawagoe, Saitama 350, Japan. *Deceased as at April 12. 1992.
[ 1,2]. SP-A is the major surfactant apoprotein and its concentration in amniotic fluid, tracheal aspirate or gastric aspirate of the newborn infant is useful for the evaluation of fetal lung maturity or the prediction of respiratory distress syndrome (RDS) of the newborn infant [3-5]. The pathogenesis of RDS is recognized as a deficiency of pulmonary surfactant, mainly due to lung immaturity. In addition, interference of surfactant function caused by inhibitors such
637 (59) Nagai et al. as meconium aspirated to the lung or proteins leaked from sera can develop similar pathophysiology caused by this quantitative deficiency. Recently, the effectiveness of exogenous pulmonary surfactant administration has been reported in cases without RDS whose respiratory failure might be due to insufficient endogenous pulmonary surfactant [61. However, little information is available about the dynamic change of endogenous pulmonary surfactant in such disorders. The aim of this study was to investigate the dynamic change of endogenous pulmonary surfactant in respiratory disorders other than classic RDS in the first week of life, and to compare the SP-A concentration of tracheal aspirate with clinical and laboratory findings.
Materials and Methods Patients and specimens A total of 30 samples were obtained from one full-term and six preterm infants who were admitted to the neonatal intensive care unit at Saitama Medical Center, Saitama Medical School, from February to December 1990. The diagnoses of these infants were as follows: pure RDS (one) and complicated RDS (three, all of whom received exogenous pulmonary surfactant therapy with surfactant-TA), transient tachypnea of the newborn (TTN; one) and congenital pneumonia (two; Table 1). All patients received mechanical ventilation soon after birth. Three of these infants were treated with conventional mechanical ventilation (CMV) and the other four infants were initially on high frequency oscillatory ventilation (HFOV), accord-
ing to the judgment of the attending doctor. The initial samples were obtained within the first 24 hr of life and subsequently every 24 hr after the initial sample was obtained. All samples were frozen at - 80°C until assayed. Parental consent was obtained for collecting all samples.
SP-A assay The two-site immunoassay with two monoclonal antibodies described by Kuroki et a1 was adopted to determine the SP-A concentration [3, 71. The tracheal aspirate sample or SP-A standard was mixed with peroxidase-labeled monoclonal antibody (PE-I0) in the test tube, and then the second antibody (PC-6) immobilized on polystyrene beads was placed in each test tube. After incubation at 37°C for 90 min, the test tube was washed, and the substrate (0.005 mol/l H,O, in 0.1 mol/l phosphate/ citrate buffer, pH 4.0) and developer solution (0.06% tetramethyl benzidine-HCl, pH 2.0) were added to each tube. After the second incubation at 37°C for 30 min, the peroxidase reaction was stopped by the addition of sulfuric acid. The absorbance was measured at 450 nm. All samples were run in duplicate. The concentration of albumin for each sample was also measured by laser nephrometry. The concentration of SP-A was standardized by dividing the SP-A content by the albumin present in the aspirate. The results were evaluated against clinical condition and laboratory data, such as arterial-alveolar oxygen tension ratio (a/APo2) and ventilatory index (Fio2 MAP/Pao,). +
Table 1. Clinical characteristics of the study group Case
Gestational age
Birthweight (g)
Diagnosis
Mode of ventilation
A B C D
26 weeks, 6 days 26 weeks, 5 days 26 weeks, 3 days 33 weeks, 5 days
1020 750 836 1278
CMV HFOV + CMV CMV + HFOV HFOV + CMV
E F G
33 weeks, 3 days 24 weeks, 1 days 38 weeks, 5 days
1228 618 2758
RDS (IV) RDS (IV) t PDA RDS (IV) t PDA RDS (IV) Pena-Shokeir I syndrome TTN + PDA Pneumonia Pneumonia PPHN
CMV CMV HFOV + CMV
Acta Paediatr Jpn
Surfactant apoprotein in tracheal aspirate (60)638
Results The SP-Nalbumin ratios in two preterm infants diagnosed with RDS by clinical features and chest X-ray findings are presented in Fig. 1. The microbubble stability test [8j performed with gastric aspirate obtained at birth was 'weak' in both cases, and the concentrations of SP-A in the same specimens were 0.1 &ml in case A and 0.3 &mi in case B, respectively. The SP-A/ albumin ratios of initial tracheal aspirates were low in both cases (0.1 @mg in case A and 2.7 pglmg in case B, respectively). In case A, the SP-Alalbumin ratio rose to 12.5pgImg on the second day of life along with improvements in the a/AP02 and ventilatory index. In case B, HFOV improved his oxygenation without surfactant replacement therapy and the SP-A/ albumin ratio increased to 21.0pg/mg on the second day of life. However, his condition again deteriorated with the white-out appearance of
chest X-ray. Surfactant-TA was given immediately with the change in the mode of mechanical ventilation from HFOV to CMV. Subsequently, pulmonary hemorrhage due to patent ductus arteriosus (PDA) was confirmed by echocardiography and a temporary decrease in aIAPo2 and the SP-Nalbumin ratio was observed. Single use of mefenamic acid was effective for the closure of the ductus, and the SP-Alalbumin ratio increased again along with the improvement in a/APOZon the fifth day of life. In contrast, in two cases with RDS the elevation of the SP-Nalbumin ratio was not observed (Fig. 2). In case C , the complication of severe pulmonary hemorrhage due to PDA was evident on the second day of life. Switching the ventilation mode from CMV to HFOV improved a/AP02 and the ventilatory index, but the SP-A/ albumin ratio remained less than 5 .Opglmg. Case D had RDS accompanied by Pena-Shokeir I syndrome. She commenced HFOV followed by
-5
A . G A 2 6 w 6 d . B W 1020g,RDS(IV)
5
.D
C GA 25 w
3 d. BW 836 g. RDS ( I V ) + PDA HFOV
CMV
'
S-TA
-t
P0A.rPdmon.r~
CMV
Wmorrhq.
Age (days) Age (days)
D GA 33 w 5 d. BW 1278 g, RDS (IV)+ Pena-Shokeir I synd
-
B GA 26 w 5 d . BW 750 g , ROS ( I V ) + POA
-c
wov
c
CUV
s-ll
c
.
wov
-.
CMV
//
5
0 12
OM
03
2
02
0
1
2
3
4
5
6
Age (days)
Age (days)
Fig. I :
Sequential changes in SP-Nalbumin ratio (Ua/APo2 ), (0-0) and ventilatory index (A-Q) of the infants with RDS. Case B developed pulmonary hemorrhage due to PDA, and a transient decrease of SP-Nalbumin ratio was observed in the course of PDA. S-TA, surfactant-TA.
Vol. 34 No. 6 December 1992
Fig. 2: Sequential changes in SP-A/albumin ratio, a/APo, and ventilatory index (VI) of the infants with complicated respiratory distress syndrome. Case C was complicated with severe pulmonary hemorrhage on the second day of life and case D had Pena-Shokeir I syndrome. Neither case showed an elevation of SP-Nalbumin ratio in the first 4 days of life.
639 (61) Nagai et al.
the administration of surfactant-TA, but her clinical condition remained unstable because of respiratory tract infection. During the first 4 days of life the SP-Nalbumin ratio remained less than 10.0 pg/mg. A case of transient tachypnea of the newborn is presented in Fig. 3. A systolic ejection murmur was audible on the second day of life with cardiomegaly and pulmonary congestion on the chest X-ray. Doppler echocardiography indicated the presence of a large left-to-right shunt through the ductus arteriosus. He responded to mefenamic acid and was weaned from the respirator on the seventh day of life. The SP-A/ albumin ratio was 23.4 pglmg on admission and increased to 77.3 pglmg on the third day of life. No significant decrease of SP-Nalbumin ratio was detected thereafter. Two infants with congenital pneumonia are presented in Fig. 4. Both were treated with surfactant-TA in the hope of removing the secretion and debris from the respiratory tract. Case F, an extremely low birthweight infant, showed an elevation of the SP-Nalbumin ratio to 34.9 pg/mg on the third day of life along with an improvement in respiratory failure. On the other hand, the term infant (case G) had marked hypoxemia in 100%O2 with an a/APo, of 0.03 and a ventilatory index of 0.870 on admission. Assisted ventilation with HFOV followed by the E : GA 33 w 3 d, BW 1228 g. TTN.PDA
*
CMV
*
F :GA 24 w 1 d. BW 61 8 g. Pneumonia
--
100
?
-.-F z c
50
1
2
3
Age (days)
G : GA 38 w 5 d. BW 2758 g. Pneumonia
a
C""
+ PPHN
//
9.7.
-100
?
F C
50
0
1
2
3
4
5
5 9
6
Age (days)
Fig. 4: Sequential changes of SP-A (0) and albumin concentrations in tracheal aspirate of the patients with congenital pneumonia. In case F, the SP-A/ albumin ratio ( G O ) elevated gradually along with the improvement of respiratory tract infection. The concentration of albumin in case G remained high in the first 3 days of life while the infection was evident. PPHN, persistent pulmonary hypertension of the newborn.
administration of surfactant-TA produced a significant recovery with the improvement of alAPo2 to 0.38 and ventilatory index to 0.034 on the first day of life. However, the SP-A/ albumin ratio remained less than 5.0pgmg during the first 3 days of life.
Discussion Pulmonary surfactant contains approximately 8% by weight of specific apoproteins. Recently, surfactant apoproteins were designated into four surfactant protein families (SP-A, B, C and D). SP-A and D are hvdroDhilic and SP-B and SP-C are hydrophobic. Among these surfactant apoproteins, SP-A is the most abundant with a molecular weight of26-38 kDa, and its localization in type I1 alveolar cells has been confirmed immunologically. Because surfactant-TA used
